Supplement Material
Extended Materials and Methods Section
Sample collection and western blots. Tissue samples were collected and frozen immediately
in liquid nitrogen. For Western blot, tissue homogenates were separated on SDSpolyacrylamide gels and transferred to PVDF nitrocellulose membranes. DDAH1 antibody was
generated as we previously described 1. DDAH2 antibody was obtained from Abcam. The
secondary antibodies were from Bio-Rad Laboratories. Antibodies against PRMT1 (protein
arginine methyltransferase 1), PRMT3 (protein arginine methyltransferase 3) are from Sigma.
eNOS antibody is from BD Biosciences. CAT (cationic amino acid transporter) and GAPDH
antibodies are from Santa Cruz Biotechnology Inc.
RT quantitative PCR. 2g of total RNA was used for reverse transcription reaction (Applied
Biosystems) followed by quantitative PCR using SYBR® Green PCR Master Mix (Applied
Biosystems). Primer pairs 5’-CAA TAG GGT CCA GCG AAT CTG C-3’ and 5’-GGG TAC AGT
GAG CTT GTC ATA ACG-3’ were used to amplify DDAH1. Primer pairs 5’- GAG CTG AGA
TCG TGG CAG ACA-3’/5’- GGG AGG GTC AGA GAG GCG TAG-3’ were used to amplify
DDAH2.
Measurement of NO production in vessel rings. Cross sections of aorta (~2mm) were
isolated and incubated in endothelial basal medium-2 (EBM-2, Cambrex) supplemented with
100 M L-arginine (Sigma), and stained with 10M NO-specific fluorescent probe 4, 5diaminofluoresceine diacetate (DAF-2 DA) dye (Sigma) at 37°C for 30 minutes. Some vessel
sections were incubated in EBM-2 media without L-arginine, and 100 M L-NAME was added
during the last 20 minutes of DAF-2 DA staining. After staining, vessel rings were washed with
1
DPBS and fluorescence intensity was recorded every 10 seconds for 5 minutes using an
Olympus FluoView 1000 confocal microscope 1.
ADMA content and NOx production in small mesenteric vessels. Mesenteric microvessels
were collected and stored in liquid nitrogen. ADMA content of small mesenteric vessels was
determined using the ELISA method 2. NOx production by these microvessels was also
determined using the colorimetric assay kit from Cayman Chemical Company.
Supplemental Data
Supplementary Table I. Anatomic and functional data of DDAH1 KO mice and wild type
controls under control conditions
Parameters
Wild type cont DDAH1 KO
Number of mice
Body weight (g)
Heart weight (mg)
Lung weight (mg)
Kidney weight (mg)
Ratio of heart weight to bodyweight (mg/g)
Ratio of lung weight to bodyweight (mg/g)
Ratio of kidney weight to bodyweight (mg/g)
8
30.2 ± 0.81
127± 5.43
159 ± 3.19
385 ± 11.2
4.2 ± 0.11
5.3 ± 0.17
12.8 ± 0.19
8
29.8 ± 1.11
133 ± 6.63
159 ± 5.16
372 ± 12.2
4.5 ± 0.09
5.4 ± 0.08
12.5 ± 0.31
8
543 ± 16.1
3.68 ± 0.20
2.20 ± 0.16
78.7 ± 1.98
1.20 ± 0.02
0.75 ± 0.02
8
529 ± 25.6
3.83 ± 0.21
2.48 ± 0.24
72.7 ± 3.62
1.21 ± 0.01
0.77 ± 0.02
Anatomic data
Cardiac functional data
Number of mice
Heart rate (beat/min)
LV end diastolic diameter (mm)
LV end systolic diameter (mm)
LV ejection fraction (%)
LV wall thickness at end systole (mm)
LV wall thickness at end diastole (mm)
Data are mean ± SE.
2
Relative DDAH2
mRNA content
Supplementary Figure I. Global-DDAH1-/- had no effect on DDAH2 mRNA expression in
tissues tested.
DDAH1+/+
DDAH1-/-
30
20
10
kidney brain lung muscle heart liver colon
3
Supplementary Figure II. Global-DDAH1-/- had no significant effect on protein expression of
eNOS, PRMT1, PRMT3, and CAT in all tissues tested.
A
brain
kidney
DDAH1
+/+
-/-
+/+
lung
-/-
+/+
-/-
eNOS
PRMT1
PRMT3
CAT
-actin
C
0.6
0.3
kidney brain lung
1.2
0.9
0.6
0.3
kidney brain lung
Relative CAT protein
content
0.9
E
D
Relative PRMT3 protein
content
Relative PRMT1 protein
content
Relative eNOS protein
content
B
1.6
1.2
0.8
0.4
kidney brain lung
4
1.6
+/+
DDAH1
DDAH1-/-
1.2
0.8
0.4
kidney brain lung
Supplementary Figure III. Total NOx production in mesenteric microvessels was significantly
decreased in DDAH1-/- mice * p<0.05.
DDAH1
Relative NOx content
1.2
DDAH1
1
*
0.8
0.6
0.4
0.2
0
5
+/+
-/-
DDAH1-/-
Systolic blood pressure
(mmHg)
Systolic blood pressure
(mmHg)
Supplementary Figure IV. L-arginine (400mg/kg, iv) infusion normalized blood pressure in
DDAH1-/- mice.
120
*
100
80
*
Vehicle
L-arginine
60
Pre
5
10 15 20
120
Wild type
100
80
Vehicle
L-arginine
60
Pre
time after infusion (min)
5
10 15 20
time after infusion (min)
6
Supplementary Figure V. Selective gene knockdown of DDAH1 but not DDAH2 caused ADMA
accumulation in cultured HUVEC.
A
B
DDAH2 siRNA
-
+
-
+
ADMA content (nM)
DDAH1 siRNA
DDAH1
DDAH2
GAPDH
7
270
240
210
180
150
*
Control siRNA
DDAH1 siRNA
DDAH2 siRNA
References
1. Hu X, Xu X, Zhu G, Atzler D, Kimoto M, Chen J, Schwedhelm E, Luneburg N, Boger RH,
Zhang P, Chen Y. Vascular Endothelial-Specific Dimethylarginine
Dimethylaminohydrolase-1-Deficient Mice Reveal That Vascular Endothelium Plays an
Important Role in Removing Asymmetric Dimethylarginine. Circulation. 2009;120:22222229.
2. Schulze F, Wesemann R, Schwedhelm E, Sydow K, Albsmeier J, Cooke JP, Böger RH.
Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay.
Clinical Chemistry and Laboratory Medicine. 2004;42:1377-1383.
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